Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca 2؉ /calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue. Molecular & Cellular Proteomics 6:283-293, 2007.Activity-dependent protein phosphorylation is one of the most ubiquitous and vital biochemical processes that contributes to the regulation and fine tuning of numerous cellular functions in many cell types. At the molecular level, the addition or removal of a phosphate residue can considerably affect the function and/or localization of proteins including enzymes, ion channels, scaffolding proteins, or signaling molecules. In the nervous system, many proteins are known to be phosphorylated in an activity-dependent fashion in discrete cellular compartments. In synaptic terminals, phosphorylation of pre-and postsynaptic proteins is required for basal neurotransmission and synaptic plasticity at both excitatory and inhibitory connections. Multiple protein kinases and phosphatases are necessary for the induction and maintenance of major forms of synaptic plasticity such as long term potentiation, long term depression, or depotentiation (1) and for cognitive functions (2, 3). For instance, phosphorylation can modulate the activity (autophosphorylation at Thr 286 ) and translocation (Thr 305/306 ) of CaMKII␣ 1 (4) or channel conductivity (Ser 831 and Ser 845 for GluR1) (5). Evidence from functional studies underlines the broad requirement for phosphorylation and the existence of many critical targets in nerve cells. For example, tyrosine phosphorylation is important for NMDA receptor gating, although it is uncle...

Post-translational modifications (PTMs) of proteins are biochemical processes required for cellular functions and signalling that occur in every sub-cellular compartment. Multiple protein PTMs exist, and are established by specific enzymes that can act in basal conditions and upon cellular activity. In the nucleus, histone proteins are subjected to numerous PTMs that together form a histone code that contributes to regulate transcriptional activity and gene expression. Despite their importance however, histone PTMs have remained poorly characterised in most tissues, in particular the brain where they are thought to be required for complex functions such as learning and memory formation. Here, we report the comprehensive identification of histone PTMs, of their combinatorial patterns, and of the rules that govern these patterns in the adult mouse brain. Based on liquid chromatography, electron transfer, and collision-induced dissociation mass spectrometry, we generated a dataset containing a total of 10,646 peptides from H1, H2A, H2B, H3, H4, and variants in the adult brain. 1475 of these peptides carried one or more PTMs, including 141 unique sites and a total of 58 novel sites not described before. We observed that these PTMs are not only classical modifications such as serine/threonine (Ser/Thr) phosphorylation, lysine (Lys) acetylation, and Lys/arginine (Arg) methylation, but also include several atypical modifications such as Ser/Thr acetylation, and Lys butyrylation, crotonylation, and propionylation. Using synthetic peptides, we validated the presence of these atypical novel PTMs in the mouse brain. The application of data-mining algorithms further revealed that histone PTMs occur in specific combinations with different ratios. Overall, the present data newly identify a specific histone code in the mouse brain and reveal its level of complexity, suggesting its potential relevance for higher-order brain functions.

Post-translational modifications (PTMs) of proteins in the adult brain are known to mark activity-dependent processes for complex brain functions such as learning and memory. Multiple PTMs occur in nerve cells, and are able to modulate proteins in different subcellular compartments. In synaptic terminals, protein phosphorylation is the primary PTM that contributes to the control of the activity and localization of synaptic proteins. In the nucleus, it can modulate histones and proteins involved with the transcriptional machinery and, in combination with other PTMs such as acetylation, methylation and ubiquitination, acts to regulate chromatin remodelling and gene expression. The combination of histone PTMs is highly complex and is known to be unique to each gene. The ensemble of PTMs in the adult brain, however, remains unknown. Here, we describe a novel proteomic approach that allows the isolation and identification of PTMs on synaptic and nuclear proteins, in particular on histones. Using subcellular fractionation, we identified 2082 unique phosphopeptides from 1062 phosphoproteins, and 196 unique PTM sites on histones H1, H2A, H2B, H3 and H4. A comparison of phosphorylation sites in synaptic and nuclear compartments, and on histones, suggests that different kinases and kinase motifs are involved. Overall, our data demonstrates the complexity of PTMs in the brain and the prevalence of histone PTMs, and reveals potentially important regulatory sites on proteins involved in synaptic plasticity and brain functions.